Virulence Proteins Research Articles

Biocontrol efficacy of native Metarhizium rileyi (Hypocreales: Clavicipitaceae) isolates against Spodoptera litura (F) (Lepidoptera: Noctuidae) and in silico effect of the secondary metabolites against the virulent proteins of the insect

Biocontrol efficacy of native Metarhizium rileyi (Hypocreales: Clavicipitaceae) isolates against Spodoptera litura (F) (Lepidoptera: Noctuidae) and in silico effect of the secondary metabolites against the virulent proteins of the insect

Identification of a Key Gain-of-Function Residue for Effector Binding by In Vitro Shuffling of Barley Mla NLR Genes.

Natural plant populations maintain high resistance (R) gene diversities that provide effective pathogen resistance; however, agricultural crops typically contain limited R gene diversity so resistance is often short-lived as pathogens evolve rapidly to evade recognition. The Mildew resistance locus A (Mla) R gene family of barley and wheat represents a rich source of natural genetic variation that is ideal for mining disease resistance specificities. Mla R genes encode immune receptor proteins of the nucleotide-binding leucine-rich repeat (NLR) class that recognise unrelated plant pathogens by binding secreted virulence proteins termed effectors. Using DNA shuffling, we generated a variant library by recombining the barley Mla7 and Mla13 genes in vitro. The variant library was cloned into yeast generating ~4,000 independent clones and was screened for interaction with corresponding barley powdery mildew effectors AVRA13 and AVRA7 using a yeast-two-hybrid assay. This yielded a number of MLA protein variants that interacted with AVRA13. Sequences of the interacting MLA variants can be clustered into three groups, all of which contain a critical residue from MLA13. While MLA13 and MLA7 differ by 30 residues across the LRR domain, the replacement of leucine to serine at this position in MLA7 is necessary and sufficient for interaction with AVRA13 in yeast and AVRA13-dependent immune signalling in planta. We have established a pipeline that evolves MLAs to recognise distinct pathogen effectors without the requirement for protein structural knowledge and the use of rational design. We suggest these findings represent a step towards evolving novel recognition capabilities rapidly in vitro.

Contrastive-learning of language embedding and biological features for cross modality encoding and effector prediction

Identifying and characterizing virulence proteins secreted by Gram-negative bacteria are fundamental for deciphering microbial pathogenicity as well as aiding the development of therapeutic strategies. Effector predictors utilizing pre-trained protein language models (PLMs) have shown sound performance by leveraging extensive evolutionary and sequential protein features. However, the accuracy and sensitivity of effector prediction remain challenging. Here, we introduce a model named Contrastive-learning of Language Embedding and Biological Features (CLEF) leveraging contrastive learning to integrate PLM representations with supplementary biological features. Biologically information is captured in learned contextualized embeddings to yield meaningful representations. With cross-modality biological features, CLEF outperforms state-of-the-art (SOTA) models in predicting type III, type IV, and type VI secreted effectors (T3SEs/T4SEs/T6SEs) in enteric pathogens. All experimentally verified effectors in Enterohemorrhagic Escherichia coli and 41 of 43 experimentally verified T3SEs of Salmonella Typhimurium are recognized. Moreover, 12 predicted T3SEs and 11 predicted T6SEs are validated by extensive experiments in Edwardsiella piscicida. Furthermore, integrating omics data via CLEF framework enhances protein representations to illustrate effector-effector interactions and determine in vivo colonization-essential genes. Collectively, CLEF provides a blueprint to bridge the gap between in silico PLM’s capacity and experimental biological information to fulfill complicated tasks.

In silico analysis of bacteriocins from Lactobacillus acidophilus membrane vesicles against Streptococcus mutans GtfB protein

Oral pathogens pose a significant global health concern, affecting 80% of the world’s population across all age groups. Among these pathogens, Streptococcus mutans stands out as a prominent threat. The rise in antibiotic resistance has limited the effectiveness of conventional antibiotics. In contrast, probiotics produce antibacterial peptides known as bacteriocins, which exhibit inhibitory effects against closely related and even unrelated bacterial species. Specifically, Lactobacillus acidophilus bacteriocins are encapsulated within membrane vesicles (MVs), allowing for long-distance transport and targeted delivery. However, no prior studies have investigated L. acidophilus membrane MV-encapsulated bacteriocins against S. mutans. In this study, we employed in-silico methods to target bacteriocins from MVs of L. acidophilus, against the GtfB virulence protein of S. mutans. Our findings indicate that Lactacin B exhibits non-toxic, non-antigenic, and non-hemolytic properties, making it a promising bioactive peptide candidate. Notably, Lactacin B forms strong interactions with GtfB in the active site, with a binding energy of −15.1 kcal/mol and four hydrogen bonds. MD simulations for 100 ns and MMPBSA assays of the complex further support the efficient binding. The fluctuation of RMSD and RMSF is minimal and corresponds to greater stability of the complex. Lactacin B interaction with gtfB mightreduce GtfB’s virulence potential, and hinder S. mutans adhesion to oral surfaces, subsequently mitigating biofilm formation and preventing dental caries. However, additional in vitro studies are necessary to validate these findings.

Investigation of Virulence-Related Markers in Atypical Strains of Toxoplasma gondii from Brazil

Toxoplasma gondii is an obligate intracellular protozoan parasite distributed worldwide that infects a wide range of warm-blooded animals, including humans. Recent studies sought to clarify the relationship between the alleles GRA15, ROP5, ROP16, ROP17, and ROP18 and the virulence of T. gondii isolates in mice. This work aims to analyze the variability of genes that express T. gondii virulence proteins of 103 strains. Most strains were virulent for mice (76/103–73.79%); within these, 30 were 100% lethal, and 46 caused a cumulative mortality range from 20% to 93%. For the GRA15 marker, most strains presenting allele 2 were non-lethal. For the ROP17 marker, allele 4 was associated with mortality, compared to allele 1. For the ROP18 marker, alleles 1 and 4 were associated with mortality, compared to alleles 2 and 3. A combined analysis of alleles showed low cumulative mortality when the strains presented alleles 3 and 1 for ROP18 and ROP16, respectively. On the other hand, allele 4 of ROP17 was a determinant for virulence when associated with ROP18 allele 3 and ROP16 allele 1. Our analysis shows that ROP18 is the primary determinant of the virulence of atypical strains in mice. Additionally, ROP17 genotyping should not be overlooked, as it has proven critical to enhance this prediction.

Indirect recognition of pathogen virulence proteins to activate plant immune receptors.

Indirect recognition of pathogen virulence proteins to activate plant immune receptors.

Novel genomic features in entomopathogenic fungus Beauveria bassiana ILB308: accessory genomic regions and putative virulence genes involved in the infection process of soybean pest Piezodorus guildinii.

Biological control methods involving entomopathogenic fungi like Beauveria bassiana have been shown to be a valuable approach in integrated pest management as an environmentally friendly alternative to control pests and pathogens. Identifying genetic determinants of pathogenicity in B. bassiana is instrumental for enhancing its virulence against insects like the resistant soybean pest Piezodorus guildinii. This study focused on comparative genomics of different B. bassiana strains and gene expression analyses to identify virulence genes in the hypervirulent strain ILB308, especially in response to infection of P. guildinii and growth on hydrocarbon HC15, a known virulence enhancer. Strain ILB308 showed the highest number of virulence-related features, such as candidate virulence proteins, effectors, small secreted proteins and biosynthetic gene clusters. ILB308 also had a high percentage of unique DNA sequences, including six accessory scaffolds. Gene expression analysis at 4 days post inoculation revealed upregulation of known virulence factors, including Tudor domain proteins, LysM motif-containing proteins, subtilisin-like proteases and novel genes encoding secreted effectors and heat-labile enterotoxins. Growth on HC15 led to the upregulation of genes associated with oxidoreductase activity related to cuticular alkane degradation and fermentation metabolism/antioxidant responses in the hemolymph. The low number of known B. bassiana virulence genes points to novel or unknown mechanisms acting on the interaction between P. guildinii and strain ILB308. The presence of accessory genomic regions and unique virulence genes in ILB308 may contribute to its higher virulence. These genes could be considered as potential targets for enhancing fungal virulence through genetic manipulation. © 2025 Society of Chemical Industry.

Swords and shields: the war between Candidatus Liberibacter asiaticus and citrus.

Citrus Huanglongbing (HLB) represents a significant threat to the citrus industry, mainly caused by the phloem-limited bacterium Candidatus Liberibacter asiaticus (CLas). In this review, we summarize recent advances in understanding the relationship between citrus and CLas, particularly examining the functions of Sec-dependent effectors (SDEs) and non-classically secreted proteins (ncSPs) in virulence, as well as their targeted interactions with citrus. We further investigate the impact of SDEs on various physiological processes, including systemic acquired resistance (SAR), reactive oxygen species (ROS) accumulation, vesicle trafficking, callose deposition, cell death, autophagy, chlorosis and flowering. Additionally, we focus on the functional research on specific disease-resistant genes in citrus and the molecular mechanisms underlying disease resistance. Finally, we discuss the existing gaps and unresolved questions regarding citrus-CLas interactions, proposing potential solutions to facilitate the development of HLB-resistant citrus varieties.

The phytoplasma SAP54 effector acts as a molecular matchmaker for leafhopper vectors by targeting plant MADS-box factor SVP.

Obligate parasites often trigger significant changes in their hosts to facilitate transmission to new hosts. The molecular mechanisms behind these extended phenotypes - where genetic information of one organism is manifested as traits in another - remain largely unclear. This study explores the role of the virulence protein SAP54, produced by parasitic phytoplasmas, in attracting leafhopper vectors. SAP54 is responsible for the induction of leaf-like flowers in phytoplasma-infected plants. However, we previously demonstrated that the insects were attracted to leaves and the leaf-like flowers were not required. Here, we made the surprising discovery that leaf exposure to leafhopper males is required for the attraction phenotype, suggesting a leaf response that distinguishes leafhopper sex in the presence of SAP54. In contrast, this phytoplasma effector alongside leafhopper females discourages further female colonization. We demonstrate that SAP54 effectively suppresses biotic stress response pathways in leaves exposed to the males. Critically, the host plant MADS-box transcription factor short vegetative phase (SVP) emerges as a key element in the female leafhopper preference for plants exposed to males, with SAP54 promoting the degradation of SVP. This preference extends to female colonization of male-exposed svp null mutant plants over those not exposed to males. Our research underscores the dual role of the phytoplasma effector SAP54 in host development alteration and vector attraction - integral to the phytoplasma life cycle. Importantly, we clarify how SAP54, by targeting SVP, heightens leaf vulnerability to leafhopper males, thus facilitating female attraction and subsequent plant colonization by the insects. SAP54 essentially acts as a molecular 'matchmaker', helping male leafhoppers more easily locate mates by degrading SVP-containing complexes in leaves. This study not only provides insights into the long reach of single parasite genes in extended phenotypes, but also opens avenues for understanding how transcription factors that regulate plant developmental processes intersect with and influence plant-insect interactions.

Computational analysis of Allium sativum compounds to identify thermolabile hemolysin inhibitors against Vibrio alginolyticus in shrimp

Vibrio alginolyticus is one of the major disease-causing bacteria in shrimp aquaculture. The widespread use of antibiotics in shrimp aquaculture to treat bacterial diseases has raised concerns about antibiotic resistance. As a result, alternative treatments, such as plant extract phytochemicals are being explored to mitigate these risks. This study aims to identify promising biologically active compounds from garlic (Allium sativum) that can inhibit the virulent protein thermolabile hemolysin of V. alginolyticus, which causes shrimp vibriosis. Various computational approaches, including molecular docking, pharmacokinetic analysis, and molecular dynamics simulation, were conducted to predict the compounds that can inhibit the phospholipase and hemolysis activities of the thermolabile hemolysin protein. Out of thirty-five compounds from A. sativum, protopine (CID 4970), gibberellin A7 (CID 92782), and gibberellic acid (CID 6466) demonstrated the strongest binding affinities, with scores of -9.4, -8.0, and -7.4 kcal/mol, respectively. Pharmacokinetic and toxicity analyses showed favorable drug-like properties for gibberellin A7 and gibberellic acid with no violations. Molecular dynamics simulations demonstrated that gibberellin A7 and gibberellic acid exhibited the highest stability over 100 nanoseconds. The investigation shows that gibberellin A7 and gibberellic acid from A. sativum have the potential to inhibit the virulent activity of thermolabile hemolysin. However, the study needs further in-vitro and in-vivo analysis to test our predicted results.

In vitro assessment of Bionectria ochroleuca metabolites: A promising approach for controlling root-knot nematode, Meloidogyne incognita

Meloidogyne incognita, a highly destructive root-knot nematode, causes substantial crop losses worldwide by infesting plant roots, which disrupts nutrient and water uptake. This pest is notoriously challenging to manage due to its broad host range and growing resistance to many chemical nematicides, emphasizing the urgent need for sustainable, eco-friendly alternatives. In this context, the current study investigated the nematicidal potential of secondary metabolites derived from the entomopathogenic fungus, Bionectria ochroleuca against M. incognita. The in vitro assays demonstrated a dose- and time-dependent inhibition of egg hatching and juvenile survival. At a crude metabolite concentration of 100%, egg hatching was reduced to 5.64% and juvenile mortality increased to 95.4% after 72 h. Gas Chromatography-Mass Spectrometry (GC-MS) analysis identified key metabolites, including palmitic acid, butanedioic acid, lactic acid, and oleic acid, which appear to inhibit nematode growth through mechanisms that impair cell membrane integrity, disrupt energy metabolism, and interfere with essential metabolic pathways. Further, metabolite enrichment analysis revealed their involvement in the biosynthetic pathways, such as unsaturated fatty acids, galactose metabolism, and phenylalanine metabolism. Molecular docking studies supported these findings by showing high binding affinities of these metabolites to virulent nematode proteins, including Cytochrome c oxidase subunit 1 and NAD(H) oxidase, suggesting interference with essential biological processes within the nematode. Overall, these findings position the metabolites of B. ochroleuca as promising candidates for managing nematode infestations, offering a potent alternative to chemical nematicides and thereby contributing to sustainable agricultural practices.

Virtual Screening for Molecular Targets of Emodin Against Red Complex Pathogens

Objective: Periodontitis is a chronic inflammatory disease affecting teeth' supporting tissues. It is caused by specific bacterial species, including Porphyromonas gingivalis (Pg), Tannerella forsythia (Tf), and Treponema denticola, known as the "red complex" group. These bacteria manipulate the immune response and promote tissue destruction, making them key players in periodontal pathogenesis. The present study aims to identify the potential molecular targets of Emodin against the red complex pathogens. Method: The interaction between the phytocompound Emodin and red complex pathogens was identified using the STITCH tool. The proteins identified were then classified into functional categories using the VICMPred. The virulent proteins identified were then subjected to Bepired prediction, which provided information about the epitopes in the virulent proteins. Finally, the subcellular location of the proteins was demonstrated with the pSORTb tool. Results: Carbamoyl-phosphate synthase is a large subunit identified as a virulence protein in Pg and Tf. DNA topoisomerase IV subunit A was found to be the common virulence protein for Pg and Td. The DNA gyrase subunit A and ATPase/histidine kinase/DNA gyrase B/HSP90 domain-containing protein were found to be identified in Td and Tf. It was the only protein predicted to be in the cytoplasmic membrane, while others were found in the cytoplasm. The four virulent proteins targeted by Emodin were found to harbor multiple epitopes. Conclusion: Emodin was found to interact with all three pathogens of the red complex group. However, further experimental validation is warranted to prove the antimicrobial effect of Emodin against periodontal pathogens.

Protease shaving of Mycobacterium tuberculosis facilitates vaccine antigen discovery and delivery of novel cargoes to the Mtb surface.

Tuberculosis (TB) is the leading cause of infectious disease death and lacks a vaccine capable of protecting adults from pulmonary TB. The bacterial surface is a critical interface that shapes host-pathogen interactions. Several knowledge gaps persist in our understanding of Mycobacterium tuberculosis (Mtb)-host interactions that may be addressed by an improved understanding of the Mtb surface proteome, including the identification of novel vaccine targets as well as developing new approaches to interrogate host-pathogen interactions. Here, we sought to expand our understanding of the Mtb surface proteome in service of these knowledge gaps by adapting protease shaving protocols for multiplexed quantitative mass spectrometry. Pairing quantitative mass spectrometry with the construction of validation strains, we revealed several novel Mtb proteins on the Mtb surface largely derived from the PE/PPE class of Mtb proteins, including PPE18, a component of a leading Mtb vaccine candidate. We next exploited the localization of PPE18 to decorate the Mtb surface with heterologous proteins. Together, these studies reveal potential novel targets for new Mtb vaccines as well as facilitate new approaches to study difficult-to-study cellular compartments during bacterial growth and infection.IMPORTANCEThe surface of a bacterial pathogen is a critical interface between the bacterium and the immune system. A better understanding of this interface would facilitate the discovery of new vaccine targets, new virulence proteins, and enable new technologies that modify the bacterial surface. In this study, we established a multiplexed and quantitative biochemical strategy to study the surface of Mycobacterium tuberculosis (Mtb) and identified new vaccine targets. We furthermore established design rules for new technologies aimed at modifying the composition of the bacterial surface. Specifically, we achieved a biological milestone that has not been rigorously reported previously, which is the successful modification of the Mtb surface with a non-native protein.

In Silico Optimization of a Streptomyces sp. Antimicrobial Peptide Against Oncobacteria in Oral Squamous Cell Carcinoma

ABSTRACTOral squamous cell carcinoma (OSCC) is a major public health concern, accounting for more than 90% of all oral malignancies. Pathogenic bacteria, such as Streptococcus mutans, Fusobacterium nucleatum, and Porphyromonas gingivalis, are closely linked to OSCC and promote chronic inflammation, a key factor in the tumor microenvironment. The increasing incidence of oral malignancies necessitates the development of antimicrobial peptides (AMPs) with enhanced anticancer activity as targeted therapeutics against bacteria to mitigate their impact on OSCC. A total of 182 Streptomyces genus‐derived AMPs were collected and analyzed in silico to determine their potent antibacterial and anticancer characteristics. Fourteen AMPs were tested for 3D structural alignment prediction and validation, and the most efficient peptide was determined to be sAMP2. A genome‐based analysis of the peptide sourced from Streptomyces globisporus C‐1027 was conducted to explore its evolutionary significance. The cell‐penetrating peptide activity of sAMP2 was optimized to make it more stable and better able to target OSCC host cellular receptors. The peptide Mut_sAMP2 was found to interact with the bacterial virulence proteins DEX of S. mutans, LPS of F. nucleatum, and KGP of P. gingivalis and with OSCC‐associated proteins (TNFα, IL‐6, IL‐8, p38, p53, E‐cadherin, Jak‐1, and PAR2) with greater binding affinity. The peptide Mut_sAMP2 treated OECM‐1 cells showed reduced cell survival by the cytotoxicity activity of IC50 5.4 μg/mL. To the best of our knowledge, our computational study is the first to significantly impact the development of new and effective cancer therapeutics from Streptomyces sp. derived AMPs with a broad spectrum of activity and potential evaluating to combat OSCC progression linked with oral oncobacteria.

In Silico Hybridization and Molecular Dynamics Simulations for the Identification of Candidate Human MicroRNAs for Inhibition of Virulent Proteins' Expression in Staphylococcus aureus.

Staphylococcus aureus is a major threat to human health, causing infections that range in severity from moderate to fatal. The rising rates of antibiotic resistance highlight the critical need for new therapeutic techniques to combat this infection. It has been recently discovered that microRNAs (miRNAs) are essential for cross-kingdom communication, especially when it comes to host-pathogen interactions. It has been demonstrated that these short noncoding RNAs control gene expression in the gut microbiota, maintaining homeostasis; dysbiosis in this system has been linked to several diseases, including cancer. Our research attempts to use this understanding to target specific bacterial species and prevent severe diseases. In particular, we look for putative human miRNAs that can attach to virulent bacterial proteins' mRNA and prevent them from being expressed. In-silico hybridization experiments were performed between 100 human miRNA sequences with varied expression levels in gram-positive bacterial infections and five virulence factor genes. In addition, these miRNAs' binding properties were investigated using molecular dynamics (MD) simulations. Our findings demonstrate that human miRNAs can target and inhibit the expression of bacterial virulent genes, thereby opening up new paths for developing innovative miRNA-based therapeutics. The implementation of MD simulations in our study not only improves the validity of our findings but also proposes a new method for constructing miRNA-based therapies against life-threatening bacterial infections.

Antibacterial Activity, Antiadherence Activity, Antioxidant Activity, and Molecular Docking of Sweetbrair Rose (Rosa Rubiginosa) Extract on Periodontopathogens: Porphyromonas Gingivalis and Aggregatibacter Actinomycetemcomitans – An In Vitro Study

ABSTRACT Introduction: Rosa rubiginosa extracts were prepared, and the antibacterial and anti-biofilm assays were investigated against two different periodontics-causing bacteria for the first time. The aim was to determine whether the extracts could restrict the growth of dental pathogens and to investigate the molecular docking between the ellagic acid of flower extracts and virulence proteins of dental pathogens. Materials and Method: Antibacterial activity of various extracts Rosa rubiginosa was studied. Anti-adherence activity and molecular docking of the antibacterial components of the extract was analysed, Result: The antibacterial activity of ethanol and methanol extracts of Rosa rubiginosa was determined using the standard agar well diffusion method. Among them, methanol extracts showed more activity against all the dental pathogens. Methanol extracts (300 μg/ml and 400 μg/ml) showed inhibitory zones of 13.6 ± 0.75 mm and 15.9 ± 0.57 mm against Porphyromonas gingivalis; Aggregatibacter actinomycetemcomitans showed inhibitory zones of about 12.9 ± 0.57 mm and 16.6 ± 0.75 mm. Minimum inhibitory concentration studies revealed that 500 μg/ml of methanol extracts inhibited the turbid growth of Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans in their broth tubes. Antiadherence test results showed that Rosa rubiginosa extracts significantly reduced the number of organisms adhering to the glass specimens against all the test cultures. The molecular docking report revealed a binding energy of about -3.57 Kcal/Mol between ellagic acid and mfa1 of Porphyromonas gingivalis. Ellagic acid and target protein cdtA of Aggregatibacter actinomycetemcomitans revealed a maximum binding energy of -4.33 Kcal/Mol. Conclusion: The findings concluded that Rosa rubiginosa extracts can be a valuable additive in natural antidental biofilm products.

Antibacterial Activity, Antiadherence Activity, Antioxidant Activity, and Molecular Docking Analysis of Arab Rose (Rosa Damascena) Extract on Periodontopathic Bacteria: Porphyromonas Gingivalis and Aggregatibacter Actinomycetemcomitans - An In vitro Study

ABSTRACT Introduction: Rosa damascena extracts were prepared, and their antibacterial and antibiofilm assays were investigated against two different periodontopathic bacteria (Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans). The aim was to determine whether the extracts could restrict the growth of these dental pathogens and to investigate the molecular docking between the myricetin of flower extracts and virulence proteins of dental pathogens. Materials and Method: The antibacterial activity of ethanol and methanol extracts of Rosa damascena was determined using the standard agar well diffusion method. Among them, methanol extracts showed more activity against dental pathogens. Methanol extracts (300 and 400 μg/mL) showed inhibitory zones of about 13.3 ± 1.05 mm and 16.3 ± 1.05 mm against Porphyromonas gingivalis; Aggregatibacter actinomycetemcomitans showed inhibitory zones of about 13.9 ± 0.57 and 15.3 ± 1.05 mm. Result: Minimum inhibitory concentration studies revealed that 400 μg/mL of methanol extracts inhibited the turbid growth in the broth tubes. Antiadherence test results showed that Rosa damascena extracts reduced the number of organisms adhering to the glass specimens against the test cultures significantly. The molecular docking report revealed −2.53 kcal/mol of binding energy between myricetin and mfa1 of Porphyromonas gingivalis. Myricetin and target protein, cdtA of Aggregatibacter actinomycetemcomitans revealed a binding energy of −3.46 kcal/mol. Conclusion: These findings conclude that Rosa damascena extracts can be considered an additive in natural antidental biofilm oral health products.

Sodium lignosulfonate inhibits multiple virulent proteins of human fungal pathogen Candida albicans.

Systemic mycoses, particularly those caused by Candida albicans, represent a serious global health concern due to rising multidrug resistance and limited treatment options. This study explores the antifungal potential of sodium lignosulfonate (LIG), a natural phenolic compound, as a multitarget therapeutic agent against various virulence proteins of C. albicans and other pathogenic Candida species. The objective of this study was to further evaluate its multiple-targeting/polypharmacological potential with plausible mode of action against C. albicans. At first, LIG was subjected to in-silico analysis to acquire preliminary knowledge about its multiple targeting potential. Subsequently, some biochemical analyses were performed to demonstrate its fungicidal activity. In-vitro analysis (plasma membrane permeation, ROS production, chitin depletion study) was performed to further validate its promising multiple-targeting/polypharmacological potential and revealed its mechanism of action. Homology modeling and docking studies revealed that LIG effectively binds to critical C. albicans proteins, including ERG1, ERG11, FKS1, CHS3, CLB2, and CEK1. The docking scores indicated strong interactions, supporting LIG's potential to inhibit multiple virulence factors With ROS production we could confirm the involvement of apoptosis. Time-kill assays confirmed the antifungal effect of LIG against C. albicans, C. glabrata, C. tropicalis, and C. parapsilosis. LIG demonstrated a >3-log10 reduction in CFU/mL, and in combination with fluconazole, it showed synergistic activity, particularly reducing CFU in C. dubliniensis by 2.5-fold compared to fluconazole alone. The chitin depletion assay has reported a decrease in levels of chitin which indicates another aspect of LIG's mode of action. This study reveals LIG as a potent and persuasive natural antifungal agent that targets multiple proteins of Candida. This revelation might impact the direction of potent antifungal agent development by aiming multiple targets of fungal pathogens simultaneously.

Mycobacterium tuberculosis Rv1048c affects the biological characteristics of recombinant Mycobacterium smegmatis

Tuberculosis is a serious, infectious, zoonotic disease caused by Mycobacterium tuberculosis. Infections are transmitted in humans and livestock via aerosols. Rv1048c is a hypothetical unknown protein in the standard strain of Mycobacterium tuberculosis H37Rv. Rv1048c exists only in pathogenic Mycobacterium tuberculosis and is highly conserved; however its function is still unclear. The recombinant Mycobacterium smegmatis strain Ms_Rv1048c, with heterologous expression of the Rv1048c gene, was constructed by using the pMV261 expression plasmid. The biological characteristics of the recombinant bacteria were studied, such as their growth pattern, drug resistance, and virulence. Expression of Rv1048c significantly reduced the growth rate of the strain, enhanced its ability to form a biofilm, and reduced its tolerance to sodium dodecyl sulfate, H2O2, and various anti-tuberculosis drugs, and reduced the viability of infected RAW264.7 macrophages. Rv1048c also significantly reduced the level of early pro-inflammatory factors in infected RAW264.7 cells. Rv1048c protein is considered to be a virulence protein that might regulate the growth of M. tuberculosis strains. The results of the present study indicate that Rv1048c plays an important role in Mycobacterial infection.

Broadly inhibitory antibodies to severe malaria virulence proteins.

Malaria pathology is driven by the accumulation of Plasmodium falciparum-infected erythrocytes in microvessels1. This process is mediated by the polymorphic erythrocyte membrane protein 1 (PfEMP1) adhesion proteins of the parasite. A subset of PfEMP1 variants that bind to human endothelial protein C receptor (EPCR) through their CIDRα1 domains is responsible for severe malaria pathogenesis2. A longstanding question is whether individual antibodies can recognize the large repertoire of circulating PfEMP1 variants. Here we describe two broadly reactive and inhibitory human monoclonal antibodies to CIDRα1. The antibodies isolated from two different individuals exhibited similar and consistent EPCR-binding inhibition of diverse CIDRα1 domains, representing five of the six subclasses of CIDRα1. Both antibodies inhibited EPCR binding of both recombinant full-length and native PfEMP1 proteins, as well as parasite sequestration in bioengineered 3D human brain microvessels under physiologically relevant flow conditions. Structural analyses of the two antibodies in complex with three different CIDRα1 antigen variants reveal similar binding mechanisms that depend on interactions with three highly conserved amino acid residues of the EPCR-binding site in CIDRα1. These broadly reactive antibodies are likely to represent a common mechanism of acquired immunity to severe malaria and offer novel insights for the design of a vaccine or treatment targeting severe malaria.